The basic tenet of this proposal is that smokeless tobacco (ST) induces buccal mucosa injury, inflammation and epithelial cell proliferation, in part, by altering local kinin metabolism. We hypothesized that exposure of the buccal mucosa to ST is associated with: 1) local generation and release of kinins that have potent proinflammatory and mitogenic effects; 2) inhibition of the membrane-bound metalloenzymes angiotensin-converting enzyme (ACE EC 3.4.15.1) and neutral endopeptidase (NEP EC 3.4.3.24.11) that are widely distributed in the buccal mucosa, and under normal physiological conditions abrogate the proinflammatory and mitogenic effects of kinins by degrading the peptides; 3) potentiation of kinin- induced proliferation of buccal mucosa epithelial cells; and 4) upregulation of bradykinin receptors on buccal mucosa epithelial cells possibly through ras transformation. The net result would be a slower turnover of kinins in the buccal mucosa with an increase in the number and/or affinity of kinin receptors on epithelial cells leading to amplification of their proinflammatory and mitogenic effects. In addition, kinin-induced edema formation and extravasation of humoral growth factors may contribute to ST-induced buccal mucosa injury, inflammation and epithelial cell proliferation. to test our hypothesis, we will use physiological, biochemical, and cell biology techniques. We chose the hamster cheek pouch as a model for the proposed studies because: 1) it is a target organ for ST; 2) the hamster cheek pouch is a well established model to study kinin metabolism; and 3) it has been used extensively for the experimental induction of oral epithelial cell dysplasia and cancer. The specific aims are to investigate: 1) whether ST increases vascular permeability in the hamster cheek pouch, and whether these effects are modulated by ACE and NEP; 2) whether ST induces kinin generation and release in the hamster cheek pouch; 3) whether ST decreases the expression and activity of ACE and NEP in the hamster cheek pouch; 4) whether ST interacts with kinins to induce buccal mucosa epithelial cell proliferation in vitro, and whether these effects are modulated by ACE and NEP; and 5) whether ST upregulates kinin receptors in cultured hamster cheek pouch epithelial cells, and whether these effects are associated with increased expression of Ki-ras mRNA. The results of the proposed studies will provide important new insights into possible mechanisms that mediate ST-induced buccal mucosa injury. In the longer term, they may provide a rationale for the development of novel diagnostic and therapeutic strategies for the management of ST-induced buccal lesions in human subjects.